Recently, medical investigations on nerve cells and investigations of the possibility of using nerve cells as electric elements have been actively pursued. When nerve cells are active, action potential is generated. The action potential is generated by the change of ion concentration inside and outside the cell membrane which is accompanied by the change of ion permeability in nerve cells. Measuring this potential change accompanied by the ion concentration change (that is, the ion current) near the nerve cells with electrodes enables the detection and investigation of nerve activities.
Conventionally, in order to measure the electrical activities of nerve cells, it is common practice to use a recording electrode comprising glass or other electrodes and a stimulating electrode comprising metal or other electrodes, insert each of them in or between cells, and measure the electrical activities of nerve cells with the recording electrode when a stimulating current (or voltage) is applied from the stimulating electrode.
In addition to this, there are many modified methods such as the so-called patch clamp method, in which a cell body is pierced with a capillary glass suction electrode, the inside of the cell body is refluxed with the liquid in the glass suction electrode, and electrical signals are emitted from this glass suction electrode to observe electric characteristics of the cell membrane.
Furthermore, a method of recording electrical activities of nerve cells is proposed separately from the inventors of this invention, which is accomplished by forming electrodes made of a conductive material such as ITO (indium tin oxide) on the surface of an insulating substrate with a diameter of 15 to 20 .mu.m, enabling culture of nerve cells on the electrodes, and applying electric stimulation to cells without piercing the cells with electrodes.
As an improvement of this method, the inventors of this invention also propose separately to form electrodes with a diameter of 20 to 200 .mu.m, so that an electric potential difference arising between the electrodes becomes smaller when constant current stimulation is applied to nerve cells. As a result, ITO is less likely to be destroyed, thereby enabling even more long-term observation.
In the above-mentioned conventional technique and its modified methods, electrodes such as glass electrodes, which have to be larger than the cells themselves, must be urged. As a result, primarily due to restrictions of space and operating accuracy, multi-point simultaneous measurements in which two or more recording electrodes are inserted simultaneously in one sample to record electrical activities of the nerve cells are extremely difficult.
In order to investigate the operation of the whole nerve circuit network, it is necessary to record many nerve cell activities simultaneously, and as the number of measuring points increases, the degree of difficulty increases, creating the problem that it is difficult to observe throughout a large number of cells.
In addition, because glass, metal, or other electrodes must be pierced into or between cells, there is another problem that the damage to the cell is serious and measurement over a long time such as extending for more than a few hours is difficult to carry out.
On the other hand, signal transmission throughout a large number of cells can be observed by using an insulating substrate formed thereon with circular (or square) electrodes made of a conductive material such as ITO with a diameter (or a side) of 15 to 20 .mu.m. However, due to the small area of the electrodes ranging from 177 .mu.m.sup.2 to 400 .mu.m.sup.2, the electrode resistance at the interface of culture solution becomes several M.OMEGA.. Since the stimulation is generally provided as constant electric current, an extremely large potential difference arises between the electrodes when the electric resistance is large. Thus, ITO is destroyed when a long-term electric stimulation is provided under such large voltage, creating the problem that it is difficult to carry out observations over a long time.
In addition, when the electrode area ranges from 400 .mu.m.sup.2 to 40000 .mu.m.sup.2, the electrode resistance at the interface of culture solution is reduced, and a potential difference arising between the electrodes becomes comparatively small. Even if the stimulating electric current was provided over a long time, destruction of ITO was not observed by a microscope. However, when a stimulating current was applied at a certain electrode and a potential change accompanied by the stimulation was recorded at other electrodes, a great change was observed in the recording waveform before and after long stimulation. In other words, the effects of the applied stimulating current on the recording waveform (that is, artifacts) were greater after long stimulation than before long stimulation. The reason for this waveform change is considered to be caused by polarization on the electrode surface. In the worst case, the electrical activities of the nerve cells were hidden by the artifacts and measurement was disabled. Furthermore, even if the artifacts are not so great, there was another problem that it becomes difficult to compare strength of nerve activities before and after long stimulation.